Project Summary/Abstract Humans are densely colonized with symbiotic bacteria ? collectively referred to as the microbiome ? that modulate physiology, for example through potent small molecules. Extensive efforts have both identified crucial roles of the microbiome in healthy human biology and linked composition of the microbiome and its associated small molecules to diseases ranging from autoimmune diseases to neurological disorders. The underlying molecular mechanisms of host-microbiome interactions, however, are largely undefined, impeding the development of therapeutic interventions for microbiome-associated diseases. Recent advances in high-throughput functional genomics methods to systematically probe the functions of human and bacterial genes, such as CRISPR-based genetic screens, now in principle enable systematic studies into the molecular mechanisms of host-microbiome interactions. To achieve their potential for microbiome biology, such systematic screening methods must be implemented and validated in model systems that recapitulate phenotypes relevant to host-microbiome interactions; once implemented, they represent powerful tools to identify genes involved in producing these phenotypes. This proposal describes the development of functional genomics approaches for primary human dendritic cells, a model system for interactions between the microbiome and the human immune system, and the application of these approaches to the identification of host receptors and physiological roles of two classes of bacterial glycolipids, capsular polysaccharides and lipopolysaccharides. Both classes are highly abundant in the microbiome and individual examples have suggested central roles for these molecules in modulating immune function. For example, individual capsular polysaccharides are known to induce differentiation of regulatory T-cells, and lipopolysaccharides are ligands for innate immune cells and elicit varying responses ranging from inflammation to immunosuppression. In this proposal, the development of new bacterial strain libraries with controlled variations in glycolipid structure is combined with functional genomics approaches to conduct structure-function analyses of the effects of glycolipids on immune cells and to identify the host receptors and signaling pathways that mediate these effects. This dissection of the mechanisms underlying glycolipid signaling at the host-microbiome interface will improve understanding of the role of the microbiome in modulating function and maturation of the immune system. The approaches described in this proposal will be similarly applicable to study the roles of other microbiome-derived molecules, human receptors implicated in microbiome diseases, or individual bacterial strains. Altogether, this proposal will both provide new insight into the biology of the microbiome and establish generally applicable approaches to decipher mechanisms of host-microbiome interactions.